Chapter 2 Radha Maharjan, Ph.D. Thesis14.139.121.106/pdf/2016/jan16/5. Ms. Radha Hiralal...

Chapter 3 Radha Maharjan, Ph.D. Thesis 2015

Materials and Methodology

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CHAPTER 3

MATERIALS & METHODOLOGY



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3.1 MATERIALS

3.1.1 ANIMALS

Adult Charles foster female rats weighting 200 +15 g were used for the study. All rats

were housed in cages and maintained in ambient temperature of 25±1C and 45.5%

relative humidity, with a photoperiod cycle of 12 h:12 h (light and dark) with access to

standard pellet food (Sai Durga Feeds and Foods, Bangalore, India) and water. Each

experiment was priorly approved by the Institutional Animal Ethics Committee,

according to the guidelines of Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Govt. of India.

3.1.2 CHEMICALS

Bovine serum albumin (BSA), Folin & Ciocalteu’s (FC) phenol reagent Nicotinamide

adenine dinucleotide reduced (NADH), Ethylenediaminetetraacetic acid (EDTA), n-

butanol, Sodium dihydrogen phosphate dehydrate (NaH2PO4.2H2O), Copper sulphate

(CuSO4.5H2O), Sodium hydroxide (NaOH), Di-potassium Hydrogen Phosphate

(K2HPO4), p-Nitrophenol (PNP), Sodium Dodecyl Sulphate (SDS), 1-chloro-

2,4dinitrobenzene (CDNB), Tris-base, Ethidium bromide, Ethylene Glycol Tetra Acetic

acid (EGTA), Trypan blue, Ammonium persulphate (APS), Tween-20, Phenyl Methane

Sulfonyl Fluoride (PMSF), Stigmasterol, para-Nitrophenylphosphate (pNPP) from SRL,

Mumbai.

Diethyl pyrocarbonate (DEPC), Agarose, Glycine, β-mercaptoethanol, Acrylamide, Bis-

acrylamide, Tetramethylethylenediamine (TEMED), Triton-X 100 from Himedia.

β-Sitosterol, Lupeol, PCR Master Mix purchased from Sigma Aldrich, India. Analytical

grade solvents like Methanol, Petroleum Ether, Toluene, Ethyl acetate, Formic acid,

Glacial Acetic acid, were obtained from Qualigens (Mumbai, India.). Precoated silica gel

aluminium plate 60 F254, (10 cm × 10 cm with 250 μm thickness was procured from

Merck, Mumbai, India. Primers were designed by PRIMER BLAST software from

National Centre for Biotechnology Information (NCBI) and purchased from Sterling

Biotech Ltd., Mumbai, India. Primary antibodies were purchased from Cell Signalling

Technology (CST) and Santa Cruz Biotechnology (SCBT). Secondary antibody was



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purchased from Merck Millipore, Germany. cDNA synthesis kit was procured from

Thermo Fisher Scientific, Mumbai, India.

3.2 METHODOLOGY

3.2.1 INDUCTION OF PCOS RAT MODEL

Charles foster virgin female rats (2-3months) exhibiting regular estrus cyclicity, weighing

180-225 g were maintained under controlled conditions of light and temperature; with

having free access to diet and water. Animals were treated orally with Letrozole (0.5

mg/kg body weight) (Kafali et al., 2004) daily for 21 days, while control group received

Carboxymethylcellulose (1% CMC) as vehicle. After 21 days of treatment, PCOS rat

model was validated by various biochemical parameters as follows:

3.2.2 ORAL GLUCOSE TOLERANCE TEST (OGTT)

(Buchanan et al., 1991)

OGTT was performed after 12 hours of fasting. The blood sample was collected in

fasting condition and following which glucose was fed (1gm/kg body weight) orally to

the rats. After glucose load, blood was periodically collected at the time interval 30’, 60’,

90’, 120’ for blood glucose estimation. Glucose was assayed at all the specified time

points by kit (Enzopak, India) based on GOD POD principle.

Glucose (GOD-POD method)

Principle:

The method is an enzyme coupled reaction for the estimation of blood glucose. Glucose

Oxidase (GOD) catalyses the oxidation of aldehyde group of glucose in the presence of

oxygen to form gluconic acid with liberation of H2O2. Peroxidase (POD) converts H2O2

into water and active oxygen which reacts with chromogen 4-amino anti-pyrine to form a

pink colored complex which can be estimated colorimetrically at 505nm.

Sample Preparation: Blood was collected in fresh vial coated with Sodium Flouride

(NaF) (6mg/ml). NaF is an anticoagulant and it inhibits glycolytic pathway. The blood

was centrifuged at 1200 g for 15 minutes for plasma separation. The plasma was

collected into fresh vials and immediately processed for glucose estimation.



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Glucose + O2 + H2O GOD

Gluconic acid + H2O2

H2O2 + 4-amino antipyrene + Phenol POD

Red colored quinol + H2O

Reagents:

i) Enzyme GOD-POD

ii) Glucose std. (100mg/ml)

iii) Chromogen

Procedure:

REAGENT BLANK STANDARD TEST

Enzyme 1.0 ml 1.0 ml 1.0 ml

Std. - 0.01 ml -

Sample - - 0.01 ml

Mix well and incubate for 5 Min at 37C. Mix & read absorbance at 505nm against

blank.

Calculation: Glucose concentration (mg/dl) = Absorbance of Sample x 100

Absorbance of STD

Unit: mg/dl

Normal Values: - Fasting: - 70-110 mg/dl

- Two hour post prandial :- up to 140 mg/dl

3.2.3 ESTRUS CYCLE

Cyclicity was checked in all rats by vaginal smear wherein saline dipped ear bud

was inserted into vagina of the rat slowly and uniform smear was made on glass slide.

These slides were observed under light microscope (10x). Each stage of the cycle was

confirmed by types of cells were observed under microscope given below.

Estrus cycle with vaginal changes:

Pro-estrus: Thickening of epithelium, Leucocytes no longer migrate through

the epithelium. Cells were small, round, nucleated epithelial.

Estrus: Outer layer of epithelial cells become cornified and sloughed into

lumen. In the beginning of phase, cell retain nuclei, but in later stage no

nuclei visible. These cells were irregular, flat and cornified.



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Metaestrus: Deeper layers of estrus epithelium line the lumen, superficial

layer become cornified & sloughed off with reduction of mitotic activity in

epithelium. Many Leucocytes & few cornified cells were seen.

Diestrus: Thinning of epithelium & leucocytes of stroma migrate through the

epithelium in the vaginal lumen. Stringy mucous with many leucocytes a few

nucleated epithelial cells are seen.

3.2.4 HORMONAL PROFILE

Sample Preparation: Blood samples were collected in fresh vials and

centrifuged at 1200 g for 15 minutes to separate serum from whole blood. Separated

serum sample then transferred into another fresh vial.

Serum was collected from all the rats used for hormonal profile. Serum hormones

levels were checked in all animal groups using ELISA kits procured from Diametra Inc,

Germany. Sensitivity of the kits was as follows: Insulin -2μIU/ml; Testosterone -0.075

ng/ml; Progesterone -0.05 ng/ml and 17 β-estradiol (8.68pg/ml) at 95% confidence limit.

The variability within run replicate is 2% for Insulin, 4.6% for Testosterone; < 5.9%

(Progesterone), < 9% (17 β-estradiol) whereas between assay variability was 6%

(Insulin), 7.5% (Testosterone), < 10.5% (Progesterone), < 10% (17 β-estradiol).

Procedure: Samples were processed according to Kit (Meril Diagnostics,

Canada) protocol. In this method, Serum samples were added [For Insulin 100μl,

Progesterone 20μl, Testosterone 25 μl, Estradiol 25 μl] in pre-coated ELISA plate. The

plate was incubated for 2 hours in case of insulin and 1hour for rest of steroids. After

incubation, the plate was washed 3 times with 300 μl wash buffer and added 100μl of

TMB (3,3',5,5'-Tetramethylbenzidine) substrate with 15 minutes incubation in dark. Then

100μl of Stop solution was added to each well to stop the reaction. Shake gently and read

at 450 nm.

3.2.5 HISTOLOGICAL ANALYSIS

Ovaries were removed and were fixed in 4% aqueous solution of formaldehyde,

often called 10% formalin. Histological examinations of ovary from all groups were



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carried wherein trans-section of 5 μm thickness cut and stained with Hematoxylin- Eosin

stain and images were taken for each group under Leica light microscope under 4 x

magnifications.

3.2.6 3 BETA- AND 17 BETA-HYDROXY STEROID DEHYDROGENASE

(Shivanandappa and Venkatesh, 1997)

Principle:

3β HSD converts DHEA to Androstenedione & 17β HSD converts 17 Estradiol to

Estrone. In both reactions, NAD is reduced to NADH, which is coupled to the Iodo Nitro

Tetrazolium Chloride (INT) and gives formazan, a pink coloured chromophore that

absorbs light at 490nm.

DHEA + NAD 3

β HSD

Androstenedione + NADH

17Estradiol + NAD 17 β HSD

Estradiol + NADH

Sample Preparation: Make 10% homogenate of tissues (ovary, uterus, adrenal,

hypothalamus, pituitary) in 0.1 M Tris-Cl buffer (pH- 7.8) and centrifuge 10,200 g for 30

min at 4ºC. Supernatant was used as a source of enzyme assay.

Reagents:

i) Phthalate buffer (50mM, pH-3.0): To 51 ml of N/10 (0.1 N) HCl add 2.5 ml of Tween

20. Mix well & add 2.55 g Potassium Hydrogen Phthalate to the mixture. Adjust to pH

3.0 and make up to volume to 250 ml with distilled water.

ii) 0.1M Tris-HCl buffer (pH-7.8)

iii) 0.5mM NAD (Freshly Prepared)

iv) Colour reagent: Dissolve 40 mg INT in 50 ml of distilled water and add 0.5 of Tween

20. For the Standard solution add 10 mg PMS. (Store in Dark)

v) Substrate: (Freshly Prepared)

DHEA (1mM): Dissolve 1mg of DHEA in 50-100 μl of DMFO. Make up the

volume to 35 ml with distilled water.

17Estradiol (1mM): Dissolve 1 mg of Estradiol in 50-100 μl of 100% Ethanol.

Make up the volume to 36.8 ml with distilled water.

vi) Standard (Range: 25 -175 nmoles): 1mM stock NADH solution

Reagents I, II and IV are stored at 4ºC and are stable for 3 months.



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Procedure:

Reagents TEST BLANK

1 0.1 M Tris-Cl (pH-7.8) 1.0 ml 1.0 ml

2 NAD 1.0 ml 1.0 ml

3 Substrate (Estradiol/Testosterone) 1.0 ml 1.0 ml

4 INT 0.1 ml 0.1 ml

5 Enzyme source 0.1 ml 0.1 ml

Incubate at 37C for 1 hour

6 Phalate buffer (pH-3.0) 2.0 ml 2.0 ml

Mix well the tubes and read at 490 nm.

Calculation: Calculation was done according to the standard graph.

Unit: nmoles of NADH formed/min/mg protein.

3.2.7 PROTEIN ESTIMATION

(Lowry et al., 1951)

Principle: Tyrosine & Tryptophan present in tissue homogenates react with Folin

Ciocalteau (FC reagent) to give blue colour complex. The color formed due to the

reaction of alkaline copper with protein & reduction of phospho-molybdate by the

protein. The intensity of colour depends on the amount of aromatic acids present, whose

absorbance is read at 660 nm.

Reagents:

i) Lowry’s Reagent:

Reagent A—2 % Sodium Carbonate dissolved in 100 ml of 0.01N NaOH

Reagent B—1 % Copper Sulfate

Reagent C—2 % Sodium Potassium Tartarate in 100 ml of Distilled Water

To make 100 ml of Lowry’s reagent (Reagent-D) of reagent A with 1 ml of reagent C &

1 ml of reagent B.

ii) Folin Ciocalteau reagent (FC reagent): [Freshly prepared]

Mix equal volume of FC reagent and D/W (1:1 ratio).



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iii) BSA: [Range: 5-50µg] Dissolve BSA (10 mg) in 10 ml of D/W (Standard

concentration 1mg/ml).

Procedure:

REAGENT BLANK TEST

Aliquot - 0.01 ml

Distilled Water 0.1 ml 0.09 ml

Reagent-D 1.0 ml 1.0 ml

Keep at Room Temperature for 10 minutes

FC Reagent 0.1 ml 0.1 ml

Mix well & Keep at Room Temperature for 20 minutes (In Dark)

Read absorbance at 660 nm


Unit: mg/ml

3.2.8 ALKALINE PHOSPHATASE (ALP)

(Bowers and McComb, 1975)

Principle: The enzyme reacts with p-Nitrophenyl Phosphate (PNNP) & converts it into

p-nitrophenol (PNP), which is yellow in colour. PNP in alkaline medium gives bright

yellow which can be measured at 405nm.

Sample preparation: Implanted area from uterus was excised and 10 % homogenate

was made in glycine buffer and centrifuged at 900 g for 10 min. Supernatant was used as

enzyme source. Serum was also source of enzyme for Toxicological parameters.

Reagents:

1. 0.4% p-Nitrophenyl phosphate (PNPP) in distilled water.

2. 0.05 M NaOH

3. Glycine buffered substrate: Mix 5.7g of glycine and 0.095 g of anhydrous MgCl2 in

750 ml of distilled water, add 85ml of 1N NaOH and make upto 1L.

4. Working substrate: Mix equal volume of glycine buffer substrate and stock PNPP.

Adjust pH to 10.3-10.4.

5. Standard PNP solution (Range: 0.04-0.16 µmole): stock 1mM in 0.05N NaOH

(Prepared freshly)



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Procedure:

TEST BLANK

1. Buffered substrate 1.0 ml -

Place tubes in water bath at 37C for 5 minutes.

2. Sample (Tissue/Serum) 0.05 ml -

Incubate at 37C for 39 minutes.

3. 0.05 N NaOH 10 ml 10 ml

Read at 405 nm.


Unit: μmoles of PNP formed/min/mg of protein

3.2.9 CATHEPSIN– D

(Anson, 1937)

Principle:

Cathepsin D is a lysosomal endopeptidase and known to have role in physiological &

pathological breakdown of intracellular & extracellular protein. Cathepsin – D activity is

measured by degradation of Haemoglobin (Hb) which leads to liberation of acid soluble

tyrosine & tryptophan containing peptides. These peptides are quantitatively measured by

sensitive reaction of Folin’s Ciocalteau reagent & giving the absorbance at 660 nm.

Sample preparation: Implanted area from uterus was excised and 10 % homogenate was

made in Acetate buffer and centrifuged at 100 g for 5 min. The supernatant was separated

in a fresh vial and centrifuge at 10,200 g for 30 min. The supernatant was again

centrifuged at 10,200 g for 30 min. Supernatant was separated in fresh vial and used as

enzyme source.

Reagents:

i. Acetate Buffer (pH- 3.5):

-0.1 M Acetic acid

- 0.1 M Sodium acetate

ii. Haemoglobin (Hb) Substrate solution (10g/100 ml)

iii. 0.3 M TCA

iv. 0.5 M NaOH



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v. Folin Ciaceau’s reagent: (1:2) dilution

vi. Std. Solution: 1mM tyrosine in 2.5 %TCA. Use it after 10-20 minutes after it is

dissolved properly.

Standard: Standard graph for Cathepsin D was prepared in range 4-40 µmole

TEST BLANK

1. Sample 0.2 ml -

2. Distilled water - 0.2 ml

3. Substrate 0.1 ml 0.1 ml

Shake vigorously and keep at 37C for 10 minutes

4. 0.3 M TCA 2.0 ml 2.0 ml

Shake and stand tubes at RT for 30 minutes and filter through Whatman filter paper

no.3

5. Filtrate 1.0 ml 1.0 ml

6. 0.5 M NaOH 2.0 ml 2.0 ml

7. FC reagent 0.6 ml 0.6 ml

Mix well and wait for 5 minutes & Read at 750 nm

Calculation : Calculation was done according to the standard graph.

Units : μmole of tyrosine formed /min/ mg protein

3.2.10 UDP-GLUCURONYL TRANSFERASE:

(Gorski and Kasper, 1977)

Principle: UDP-Glucoronyl transferase catalyses transfer of D-glucoronic acid from

UDP α- glucoronic acid to a compound with inversion at the C-1 carbon of the sugar to

yield the β-glucoronide. The degree of conjugation was based on the disappearance of p-

nitrophenol absorbance in the visible region. The ratio of glucoronide formation was

calculated directly from the absorbance change.

Sample Preparation: 10% homogenate of liver tissue was made in 0.25 M sucrose

solution. and centrifuged at 100 g for 5 min.. The supernatant was separated in a fresh



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vial and centrifuged at 10,200 g for 30 min. The supernatant was again centrifuged at

10,200 g for 30 min. The supernatant was separated in fresh vial and used as enzyme

source.

Reagents:

i) 0.4 M Phosphate Buffer (pH-7.4)

ii) 40mM MgCl2

iii) 1.6 mM p-Nitrophenol (PNPP)

iv) 20 mM UDP-glucoronic acid

v) 0.2 N TCA

vi) 0.5 N NaoH

vii) Standard PNP (p-Nitrophenol) solution (Range: 20-200 μmoles): 1mM stock

solution

Reagent Test Blank

Buffer 0.09 ml 0.130 ml

UDPGA 0.02 ml -

Mgcl2 0.02 ml .02 ml

PNPP 0.02 ml -

Enzyme 0.05 ml 0.05 ml

Incubate 37ºC for 10 minutes

TCA 0.2 ml 0.2 ml

Centrifuge at 2000 g for 20 minutes and take 0.250 ml of supernatant

0.5M NaOH 0.750 ml 0.750 ml

Measure absorbance at 450 nm

Calculation : Calculation was done according to the standard graph

Unit: μmoles of PNP conjugated/min/mg protein

3.2.11 HYDROXY STEROID OXIDOREDUCTASE (HSOR)

Principle:

17β HSD converts 17β -Estradiol to Estrone. In both reactions, NAD is reduced to

NADH, which is coupled to the Iodo Nitro Tetrazolium Chloride (INT) and gives



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formazan, a pink coloured chromophore that absorbs light at 490 nm.

Sample Preparation: 10% homogenate of liver was made in 0.25 M sucrose solution

and centrifuge at 100 g for 5 minutes and the supernatant was separated. The supernatant

was again centrifuged at 10,200 g for 30 min at 4ºC. Supernatant was use as a source of

enzyme assay.

Reagents:

i) Phthalate buffer (50mM, pH-3.0): To 51 ml of N/10 (0.1 N) HCl add 2.5 ml of Tween

20. Mix well & add 2.55 gm Potassium Hydrogen Phthalate to the mixture. Adjust to pH

3.0 and make up to volume to 250 ml with distilled water.

ii) 0.1M Tris-HCl buffer (pH-7.8)

iii) 0.5mM NAD (Freshly Prepared)

iv)Colour reagent: Dissolve 40 mg INT in 50 ml of Distilled Water & to which, add 0.5

of Tween 20. For the Standard solution add 10 mg PMS. (Store in Dark)

v) Substrate: (Freshly Prepared): 17β- Estradiol (1mM): Dissolve 1 mg of Estradiol in 50-

100 μl of 100% Ethanol. Make up the volume to 36.8 ml with distilled water.

vi) Standard solution (Range: 25- 175 nmoles): 1 mM stock solution of NADH

Reagents I, II and IV are stored at 4ºC and are stable for 3 months.

Procedure:

Reagents TEST BLANK

1 0.1 M Tris-Cl (pH-7.8) 1.0 ml 1.0 ml

2 NAD 1.0 ml 1.0 ml

3 Substrate (Estradiol) 1.0 ml 1.0 ml

4 INT 0.1 ml 0.1 ml

5 Enzyme source 0.1 ml 0.1 ml

Incubate at 37C for 1 hr

6 Phalate buffer (pH-3.0) 2.0 ml 2.0 ml

Mix well the tubes and read at 490 nm.

Calculation : Calculation was done according to the standard graph

Unit: nmol of NADH formed/min/mg protein.



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3.2.12 GLUTATHIONE-S-TRANSFERASE (GST)

(Habig et al., 1974)

Principle: The reaction is measured by observing the conjugation of 1-chloro, 2,4-

dinitrobenzene (CDNB) with reduced glutathione (GSH). This is analysed by an increase

in absorbance at 340nm. One unit of enzyme will conjugate 10.0 nmol of CDNB with

reduced glutathione per minute at 25C.

Sample Preparation: 10% homogenate of liver tissue was made in 0.25 M sucrose

solution. and centrifuged at 100 g for 5 min.. The supernatant was separated in a fresh

vial and centrifuged at 10,200 g for 30 min. The supernatant was again centrifuged at

10,200 g for 30 min. The supernatant was separated in fresh vial and used as enzyme

source. Serum was also source of enzyme for toxicological parameters.

Reagent:

i) 1mM Na2EDTA in 0.1 M Sodium phosphate buffer

ii) 20mM GSH

iii) 20mM CDNB (1-chloro-2,4 Di nitro benzene) dissolved in 100% ethanol

REAGENTS VOLUME

1 Buffer 0.850 ml

2 20 mM Reduced glutathione 0.050 ml

3 20 mM CDNB 0.050 ml

Auto zero & reaction was initiated by adding enzyme

4 Sample 0.050 ml

The increase in absorbance was monitored for about 2 minutes at 340 nm.

The enzymes activity was calculated using the extinction coefficient. E412=9.6 (mmol-

1cm

-1)

Change in absorbance (DA340)/minute, in the linear range of the plot, for the sample and

for the blank using the following equation:

(DA340)/min = A340(final read) - A340(initial read)

reaction time (min.)



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GST activity = ∆A340 min-1 x dilution factor

0.0096 µmol-1 cm-1 x mg of protein

Unit: Amount of Enzyme that catalyzes the conjugation of 1 μmole of CDNB per min per

mg protein.

3.2.13 LIPID PROFILE

3.2.13.1 TRIGLYCERIDES

Principle:

Lipoprotein lipase hydrolyses triglycerides sequentially into Di and Monoglycerides

and finally to glycerol. Glycerol Kinase (GK), using ATP as phosphate source, converts

Glycerol liberated to Glycerol-3-Phosphate (G-3-Phosphate). G-3-Phosphate Oxidase

(GPO) oxidizes G-3-Phosphate formed to Dihydroxy acetone phosphate (DHAP) and

hydrogen peroxide is formed. Peroxidase (POD) uses the hydrogen peroxide formed, to

oxidize 4- minoantipyrine and DHBS (3,5 Dicloro-2-hydroxy benzene sulphate) to form

a red coloured complex. The absorbance of the coloured complex is measured at 520nm.

Triglycerides + H2O Lipase

Glycerol + fatty acids

Glycerol + ATP Glycerol-3-Phosphate + ADP

Glyccerol-3-Phosphate + O2 GPO

Dihydroxyacetone phosphate + H2O2

H2O2 + 4-Aminoantipyrine + DHBS POD

Quinoneimine + H2O

Sample Preparation: Blood was collected in fresh vial without any anticoagulant and

centrifuged at 1200 g for 15 minutes. The clear supernatant comprising of serum was

transferred into fresh vial and used for the estimation.

Reagents:

i) Lipase Reagent

ii) Triglycerides Standard (200mg/dl)

iii) Triglycerides (buffer)

REAGENT BLANK STD TEST

Working Reagent 1.0 ml 1.0 ml 1.0 ml

Std. - 0.02 ml -

Sample - - 0.02 ml



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Mix and incubate at 37C for 5minutes and read absorbance of test and standard against

reagent blank at 520nm (Green filter).

Calculation:

Triglycerides (mg/dl) = Abs of Sample X 200

Abs of Standard

Unit: mg/dl ; Normal Values: Females: 45 -145mg/dl

3.2.13.2 CHOLESTEROL

Principle:

Cholesterol esters are hydrolyzed to free cholesterol by cholesterol esterase (CE). Free

cholesterol is then oxidized by cholesterol oxidase (CO) to cholesten-4-en-3-one. H2O2

is also produced simultaneously which reacts with 4-aminoantipyrine and phenol in the

presence of peroxidase and forms pink-colored quinonemine dye which is read at 505nm.

Cholesterol esters CE

Cholesterol + fatty acids

Cholesterol + O2 CO

Cholesterol-4-en-One + H2O2

2 H2O2 + 4-AAP POD

Quinoneimine dye + 4H2O.




Reagents:

i) Cholesterol L (ready to use)

ii) Cholesterol Standard (200mg/dl)

Procedure:



Std. - 0.01 ml -

Sample - - 0.01 ml

Mix and incubate at 37C for 10 minutes and read absorbance of test and standard against

reagent blank at 520nm at room temperature.



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Calculation:

Cholesterol concentration (mg/dl) = Abs of Sample X 200

Abs of Standard

Unit: mg/dl

Normal Values: 100 - 250mg/dl.

3.2.13.3 HDL-CHOLESTEROL

Principle:

High density lipoproteins (HDL) are separated from other lipoprotein fractions by

treating serum with phosphotungstic acid and magnesium chloride. HDL remains in

solution while all other lipoprotein fractions are precipitated; cholesterol content of which

is estimated by enzymatic method.

Serum + Phosphotungstic acid (PTA) Supernatant + Precipitates (other fractions).




Reagents:

i) HDL – Cholesterol (Precipitating Reagent)

ii) HDL – Cholesterol standard (50mg/dl)

iii) Standard- 50 mg/dl

Procedure:



Std. - 0.05ml -

Sample - - 0.05 ml

Mix well and incubate at 37C for 10minutes and read absorbance of test and standard

against reagent blank at 520nm.

Calculation:

Serum HDL Cholesterol (mg/dl) = Abs of Sample X 50 (Dilution Factor) X 2

Abs of Standard



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Unit: mg/dl

Normal Values:

Females: - 45 -65mg/dl

3.2.13.4 LDL CHOLESTEROL

Estimated by calculation:

LDL Cholesterol = [Total Cholesterol - HDL Cholesterol – Triglycerides/5] mg/dl

3.2.14 SERUM GLUTAMATE PYRUVATE-TRANSAMINASE (SGPT) ACTIVITY

(Reitman and Frankel, 1957)

Principle: Glutamate pyruvate transaminase transfers amino group from alanine to -

keto glutarate (-KG) & converts it into pyruvate. Thus formed is reacted with 2, 4 Di

Nitro Phenyl Hydrazine (DNPH). The resulting hydrazone of pyruvate is highly colored

and its absorbance at 540 nm is proportional to GPT activity.

L-Alanine + -keto gluterate SGPT Pyruvate + Glutamate

Pyruvate + DNPH Brown colour in alkaline medium

Sample Preparation: Blood was collected in fresh vial and allowed to stand for half n

hour. Later, samples were centrifuged at 1200 g for 15 minutes. The clear supernatant

comprising of serum was transferred into fresh vial and used for the estimation.

Reagents:

I. SGPT Substrate: 1.78 g of alanine, 30 mg of -keto glutaric acid, 20 ml of 1N

NaOH in phosphate buffer. The pH was adjusted to 7.4 and finally the volume was

made up to 100 ml with distilled water.

II. DNPH Reagent: 20 mg of dinitrophenylhydrazine in 8.5 ml of conc. HCl. Final

volume was made up to 100 ml with distilled water.

III. 0.4 N NaOH

IV. 44 mg/dl Sodium Pyruvate std. (Range: 22-110g)



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Procedure:

Test Blank

1. Serum 0.05 ml 0.05 ml

2. Substrate 0.25 ml -

3. Distilled Water - 0.25 ml

Incubate at 37C for 30 min

4. DNPH 0.25 ml 0.25 ml

Incubate at RT for 20 min

5. 0.4M NaOH 2.5 ml 2.5 ml

Read at 540 nm

Normal Range: 6-72 IU/dl

SGPT = O.D. x1000 x sample dilution

Slope x 30 (Incubation time)

Unit: μmoles of pyruvate formed/min/lit

3.2.15 SERUM CREATININE

(Bonsnes and Taussky, 1945)

Principle: Creatinine reacts with picric acid in alkaline medium to form a reddish

yellow complex, intensity of which is directly proportional to the concentration of

creatinine in sample and can be measured at 520 nm.

Sample Preparation: Blood was collected in fresh vial and allowed to stand for half an

hour. Later, samples were and centrifuged at 1200 g for 15 minutes. The clear

supernatant comprising of serum was transferred into fresh vial.1.5 ml of Picric acid was

added to 0.5 ml of Serum and then centrifuged at 3000 g for 15 minutes. Supernatant

used for the assay.

Reagents:

I. Picric acid : 0.91gm/dl (40mM)

II. 0.75 M NaOH

III. Working Creatinine Std: (10-50 g) in distilled water.



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Procedure:

Reagent TEST BLANK

1. Supernatant 1.0 ml -

2. Distilled Water 1.0 ml 2.0

3. Picric acid 1.0 ml 1.0 ml

4. 0.75 M NaOH 1.0 ml 1.0 ml

Read at 520 nm.

Creatinine (mg/dl) = O.D. x sample dilution x 100

Slope

3.2.16 GRANULOSA CELL ISOLATION

(Campbell, 1979)

Sample: Animals were sacrificed in Proestrus stage of estrus cyclicity and the ovaries

were excised and placed in tubes containing cold sterile HBSS.

Reagent:

i) Hank’s Balanced Salt Solution (HBSS)[250 ml]:

-2 g NaCl

- 0.1 g KCl

-0.015 g Na2HPO4

-0.015g KH2PO4

-0.0625 g NaHCO3

-0.25 glucose

Mixture was dissolved in minimum volume. Adjust pH at 7.2 and later volume

was made up to 250 ml.

ii) EGTA (6.8mM) containing BSA (0.2%) solution

iii) 0.5M Sucrose with 1.8mM

iv) HBSS containing EGTA (2mM)

v) Trypan Blue (0.18%)



84

Procedure:

i) Ovaries were centrifuged at 100 g for 5 minutes to remove fat in 2 ml of HBSS

solution at 4C.

ii) Ovaries were suspended in 2 ml of EGTA solution and incubated for 15 minutes

at 37C and later centrifuge at 100 g for 5 minutes.

iii) These ovaries were suspended in 2 ml of hypertonic sucrose solution and

incubated for 5 minutes in ice and centrifuge at 100 g for 5 minutes.

iv) Ovaries were expressed for cells using blunt forceps in 2 ml of HBSS solution in

cold condition. After expressing cells, cells were again centrifuged at 1500 rpm

for 8 minutes at 4C.

v) Cells were suspended in 2 ml of HBSS containing EGTA & centrifuge at 100 g

for 5 minutes.

vi) Pellet was washed with HBSS solution 3-4 times to remove fat and cell debris.

vii) Final pellet was suspended in 100-200 μl of HBSS solution.

viii) 10 μl of cells were stained with Trypan blue & counted in16 small squares of 4

corner squares of the Hematocytometer.

ix) No. Of cells were calculated by taking the average no. of cells presented per

square.

x) No. Of cells= Avg of no. of cells in all 4 Squares X 104 X dilution Factor

Expression Unit= No. of cells/ mg ovary.

Sonication protocol: Ampliture 45 %, Total Time : 20 sec, On 2 sec, Off 2 sec

3.2.17 RNA ISOLATION BY TRIZOL METHOD

1. For 50-100 mg of tissue, 1.0 ml of TRIZOL reagent was added and the tissues

were homogenized in cold condition.

2. Homogenized sample was incubated at room temperature for 5 minutes.

3. 200 µL of chloroform (per 1mL Trizol) was added and shaken vigorously for 15

seconds and incubated at room temperature for 3 minutes.

4. Samples were centrifuged at 1250 g at 4C for 15 minutes to separate phases.

5. Upper (clear) aqueous layer was transferred to a fresh tube.



85

6. 0.5mL of isopropanol was added to the aqueous layer, mixed gently and incubated

in -20C for 1 hr.

7. The samples were centrifuged at 1250 g at 4C for 15 minutes to pellet RNA.

8. The supernatant was carefully removed and 1ml of 75% DEPC-ethanol was added

to wash the pellet thoroughly.

9. Samples were centrifuged at 7500 g at 4C for 5 minutes to re-pellet RNA.

10. Carefully remove the supernatant and air-dry the pellet at room temperature for 5-

10 minutes.

11. Dissolve the pellet in sterile DEPC distilled water (10-100uL, depending on yield)

by gentle pipetting and quantitate the concentration by 260/280 nm ratio.

3.2.18 RT PCR (REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION)

All reagents were thawed and stored on ice.

RNAse and DNAse free work environment was maintained.

The individual reagents were thoroughly mixed and spinned down.

i) cDNA preparation:

RNA 2µL (2μg) was added into fresh PCR tube along with 3 µL DEPC-treated

water. It was incubated at 65C for 10 minutes and kept in ice. To that,

3µL of 5X 1st strand buffer was added along with 1.0µL dNTPs, 0.5µL oligo dT

primer, 0.5µL reverse transcriptase and incubated at 42C for 45 minutes. The cDNA

was stored in -20C for further use.

ii) PCR conditions:

1µL of cDNA (transcribed from 2 μg of RNA) was added to fresh PCR tube and

5µL Ready to use Master Mix was added along with 1µL dNTPs, 1µL forward

primer, 1µL reverse primer, 1µL sterile distilled water. Mix the tubes in microfuge

and PCR program appropriate to the primers was run.



86

iii) PCR program:

iv) Gel Electrophoresis: Run the PCR amplicon on 1.2 % agarose gel with 100

bp ladder.

v) 50 X TAE Buffer [100 ml]

Tris-base 40mM - 24.2 g

Glacial acetic acid - 5.71 ml

0.5 M EDTA -10 ml

Make upto 70 ml with distilled water, set the pH 8.4 and Make upto volume 100 ml

with distilled water.

vi) 5X TBE Buffer [200 ml]

Tris base (44mM)- 10.8 g

Boric acid - 5.5 g

0.5 M EDTA - 4.0 ml

Make upto 150 ml with distilled water, set the pH 8.4 and Make upto volume 200 ml

with distilled water.

vii) RNA Loading Dye (2x)

500mM EDTA (pH-8.0)

37% Formaldehyde

20% Glycerol

1% Formamide

0.1% Bromophenol blue

10% Electrophoresis buffer

Step Time

Initial denaturation Time 94C, 2 min

Denaturation 94C, 2 min

Annealing (According to primer

Tm value

30 sec – 2 min

Extension 72C, 30 sec- 2 min

Repeat 25-35 cycles

Final extension 72C, 10 min

Hold 4C , 2 min then end the program



87

viii) 6x Loading Dye ( 1ml)

0.2 % Bromopheol buel

20 % Sucrose

ix) Primers of rat targeted genes

Targeted

Genes Primer sequence

Annealing

temperature

(0C)

Reference

Aromatase

F: 5’GCTTCTCATCGCAGAGTATCCGG 3’

R: 5’CAAGGGTAAATTCATTGGGCTTGG 3’ 60 NM_017085

FSH

F: 5’ AGGAAGAGTGCCGTTTCTGC 3’

R: 5’ GCTGTCACTATCACACTTGC 3’ 53

Schirman-

Hildesheim et

al.2008

LH

F: 5’ CTGTCCTAGCATGGTTCGAGT 3’

R: 5’ AGTTAGTGGGTGGGCATCAG 3’ 58.4

Schirman-

Hildesheim et

al.2008

FSHR

F: 5’ CTCATCAAGCGACACCAA 3’

R: 5’ GGAAAGGATTGGCACAAG 3’ 54

Cavalcante et al.

2013

LHR

F: 5’ GCTTTTACAAACCTCCCTCGG 3’

R: 5’ GCGAGATTAGAGTCGTCCCA 3’ 55 NM_012978

ER

F: 5’ CCAGTCGAGCATCACTTA 3’

R: 5’ TGGCGTCGATTGTCAGAA 3’ 53

Suzuki M and

Nishihara, 2002

ER

F: 5’ TAGACAACCGCCATGAGT 3’

R: 5’ CACAACTGCTCCCACTAA 3’ 54

Suzuki M and

Nishihara, 2002

AR

F: 5’ GGAAGCACTGGAACATCT 3’

R: 5’ GTAGTCGCGATTCTGGTA 3’ 53

Suzuki M and

Nishihara, 2002

PR

F: 5’ CGCCCTACCTCAACTACCTG 3’

R: 5’ TTCTCAGACGACATGCTGGAC 3’ 56

NM_022847

GAPDH

F:5’CAAGGTCATCCATGACAACTTTG 3’

R:5’GTCCACCACCCTGTTGCTGTAG 3’ 58.1

NM_017008

3.2.19 SDS PAGE AND WESTERN BLOT (WB)

Sample preparation: Make 10 % homogenate in Lysis buffer, centrifuge aat 900

g for 10 minutes to pellet down cell debris then sonicate the sample twice

(Ampliture 45 %, Total Time : 20 sec, On 2 sec, Off 2 sec)



88

Sample used: Ovary, Placenta

Reagents:

i) 1.5M Tris-Cl buffer (pH-8.8)

ii) 0.5 M Tris-Cl buffer (pH-6.8)

iii) SDS Electrophoresis buffer (1L)

-3 g Tris base (25 mM)

-14 g Glycine (1.92mM)

-1 g SDS (1%)

v) Coomassive stain (100 ml):

-0.1% Commassie blue

-40% Methanol

-10% Acetic acid

- 50% distilled water

vii) 2x SDS Loading dye:

10mM Tris-Cl (pH -6.8)

` 4% SDS

0.2% Bromophenol blue

20% Glycerol

10 % β-mercaptoethanol

viii) Phosphate buffer saline (10X) (1 lit):

80g of NaCl

2.0g of KCl

14.4g of Na2HPO4

2.4g of KH2PO4

Adjust pH to 7.4, adjust volume to 1L with additional distilled H2O and sterilize

by autoclaving.

ix) PBS-T: (PBS containing 0.1 % Tween 20)[ Add 0.5 ml of tween 20 into 100

ml of 10x PBS and volume make upto 1 litre]

x) Blocking buffer/PBSMT (5% non-fat dry milk in PBS-T buffer)

xi) Transfer Buffer(10x):[Tris-base - 15.14 g, Glycine - 72.06 g]

To make 1x transfer buffer, add 100 ml of 10x Transfer buffer into 200 ml of

Methanol and volume make upto 1 litre with distilled water. Make fresh 1x

transfer buffer prior transfer and keep in cold condition.

xii) Primary antibody: Primary antibodies dilutions made according to

iv) 30% acrylamide solution (100 ml):

-Acrylamide: 30 g

- Bisacrylamide: 0.9g

vi) Lysis Buffer (For 50 ml)

- Tris-Cl (pH-7.5) 50 mM

- NaCl 200mM

- CaCl2 20mM

- Triton-X 100%

- Protease Inhibitor 1mM



89

standardized protocol in PBS-MT along with 1mM NaN3 (Final

concentration) and kept in 4C.

xiii) Secondary antibody: 1:2500 dilution in PBS-MT

Preparation of SDS PAGE gel: [20 ml]

Reagents 12% (ml) 10% (ml) 6% (ml)

Distilled water 6.6 7.9 10.6

30% Acrylamide 8.0 6.7 4.0

Running gel buffer

(pH-8.8, 1.5M)

5.0 5.0 5.0

10% SDS 0.2 0.2 0.2

10% (APS) 0.2 0.2 0.2

TEMED 0.02 0.02 0.02

Preparation of stacking gel: [10 ml]

Reagents (ml)

Distilled water 6.9

30%Acrylamide 1.66

Running gel (pH-6.8, 0.5M) 1.26

10% SDS 0.1

10% (APS) 0.1

TEMED) 0.01



90

Antibodies used in the present study:

Primary antibodies dilutions

No. Antibodies Source Dilution

1 Androgen

Recepter (AR)

Rakesh Tyagi, JNU, India 1:1000

2 StAR Stocco, Texas Tech University, Texas, USA 1:2000

3 3β-HSD Prof. Ian Mason, University of Edinburgh,

France

1:2000

4 β-actin CST, # 4967 1:10,000

5. P450arom CST , #8799 1:1000

Secondary antibody dilution

Conjugated Anti-

rabbit IgG

Puregene, GX1202E-3 1:2500

Procedure:

1) The protein molecular marker (14-97.3 kDa) and 40 μg of samples were loaded

on SDS PAGE wells and run at constant voltage (100 V, 50 current) until the dye

front of the samples reach the bottom of the gel.

2) The proteins were transferred from the gel to a nitrocellulose or PVDF membrane

in transfer unit containing transfer buffer at 4C, at 100 V for 1.5 hours.

3) After the transfer is completed, the membrane was stained with Ponceau-S to

identify the mark of the molecular weight.

4) 20 ml of blocking buffer was added and membrane was incubated for 1 hour at

room temperature with shaking.

5) The primary antibody was diluted in PBSMT as mentioned in table.

6) The membrane was incubated with the diluted antibody overnight (~16 hrs) at

4C with shaking.

7) The membrane was washed 4 times with PBST for 15 minutes each.



91

8) The secondary antibody conjugate was diluted 1:2500 in PBSMT.

9) The membrane was incubated with the diluted antibody for 1 hour at room

temperature with shaking.

10) The membrane was washed 3 times with PBST for 15 minutes each. This was

followed by 2 washes of PBS for 10 minutes each.

11) Finally, an appropriate detection method utilizing DAB (3, 3'-diaminobenzidine)

was used. Horseradish peroxidase (HRP), which is bound to the secondary

antibody, catalyzes the oxidation of DAB in the presence of peroxidase. The

oxidation product of DAB polymerizes to form a brown, sharply localized

insoluble product, which can be easily detected by visible light.

3.2.20 PREPARATION OF ALOE VERA EXTRACT

Aloe barbadensis Mill. (Voucher no. PSN 723) was compared with the specimen

(Bhatt 2486, 653, 279, JVJ 448) lying with the nationally recognized BARO Herbaria of

the Department of Botany, The M.S. University of Baroda, Vadodara, Gujarat, India. Fresh

mature Aloe vera leaves (3.5 years old) were taken and washed with water. Later, the

leaves were incised with the sterilized knife and allowed to stand for 2 hours in order to

remove the aloin. Later, the gel was removed by separating the epidermis and later gel was

sonicated to get a homogenous gel.



92

3.2.21 PREPARATION OF THE PETROLEUM ETHER EXTRACT (P1)

3.2.22 PHYTOCHEMICAL SCREENING

Major groups

of

phytochemical

constituents

Test Reagents and assay procedure Inference

Steroidal

Glycosides

Baliget test

Solution A: 1% Picric acid in

Ethanol

Solution B: 10% NaOH

Solution A and Solution B

were mixed in 1:1 ratio and

added drops by drop to 1.0 ml

of sample.

Orange to deep

red colour



93

Killer-Killani

Test

1ml of extract was mixed with

2ml of Glacial acetic acid

containing 1-2 drops of 2%

FeCl3.To this 2ml of conc.

H2SO4 was added along the

walls of the test tube.

Formation of a

brown ring at the

interphase

Phytosterols

Libermann-

bachmann

Solution: 1 ml chloroform + 1

ml acetic anhydride cooled to

0C and few drops of conc.

H2SO4 was added. This reagent

was added to the sample.

Blue, Green, Red,

Orange colour

change with time

Salkouski

Test

1mL Chloroform + 1ml conc.

H2SO4 was added to 1ml of the

sample.

Formation of Red

colour

Alkaloids Mayor’s test

Solution A: Mercuric chloride

(1.358 g) + 60 mL distilled

water.

Solution B: KI (5.0g) + 10 mL

of distilled water.

Both solutions were mixed and

the final volume was made up

to 100 mL with distilled water.

To this reagent few drops of

Sample was added.

A white or creamy

precipitate



94

Wagner’s test

Solution: Iodine (1.27g) + KI

(2 g) + 5 mL distilled water.

The volume was made up to

100 ml with distilled water.

This reagent was added by side

of the test tube to the filtrate.

Reddish brown

precipitate

Dragendroffes’s

test

Solution A: 0.85 g bismuth

nitrate in a solution of 10 mL

acetic acid + 40 ml water

Solution B: 8 g KI in 20 mL

water. Both solutions were

mixed in equal proportion and

this reagent was added to the

filtrate.

Prominent yellow

precipitates

Phenolic

Compounds

Fecl3 To the extract few mL of

distilled water and few drops

of neutral 5% FeCl3 solution

were added.

Dark Green:

Phenolic

compounds

Blue colour:

Tannins

Gelatin test

To the extract, few ml of

distilled water and 1% solution

of gelatin containing NaCl (10

%) were added.

Formation of white

precipitate

Flavonoids Lead acetate

test

To 1 ml of extract, 1ml of 10%

lead acetate was added.

Formation of

yellow precipitate



95

Terpenoids To the extract, 2ml of

chloroform was added and

shaken vigorously. The

organic phase was separated

and evaporated.2ml of conc.

H2SO4 was added and heated

for 2 minutes.

Formation of

greyish colour

Saponins Foam test To 1 ml of extract, 1ml of

distilled water was added and

warmed.

Formation of

stable foam in the

form of

honeycomb.

Carbohydrates Molish Test To 2 ml of extract, few drops

of α-naphthol were added. 1ml

of conc. H2SO4 was added in a

drop-wise manner along the

sides of the test tube

Formation of a

violet ring at the

junction of two

layers.

Protein Ninhydrin Test The extract was boiled with

2ml of 0.2% Ninhydrin

solution

Formation of

violet colour

3.2.23 QUANTIFICATION OF MAJOR PHYTOCHEMICAL CONSTITUENTS OF ALOE

VERA GEL

3.2.23.1 ESTIMATION OF TOTAL FLAVONOIDS

(Slinkard and Singleton, 1977)

Quercetin (standard) was used to make a calibration curve. Quercetin (10 mg) was

dissolved in 10 ml of 80% ethanol.Range of standard: 10-100 µg/ml.

TEST: Diluted standard solution (0.5ml) + (1.5ml) 95% ethanol.

BLANK STD SAMPLE

95% Ethanol 2.0 2.0 2.0

10% AlCl3 0.1 0.1 0.1



96

1 M CH3COOK 0.1 0.1 0.1

D/W 2.8 2.8 2.8

Incubate at room temperature for 30’ & take absorbance at 415 nm.

3.2.23.2 ESTIMATION OF TOTAL PHENOLICS

(Zheng and Wang, 2001)

p- Coumaric acid (standard) was used to make a calibration curve. p- Coumaric acid (10

mg) was dissolved in 10 ml of 95% ethanol.

Range of standard: 10-100 µg/ml.

TEST: Dilute standards upto 200µl.

BLANK STD SAMPLE

95% Ethanol 0.2 - -

Folin-Ciocalteu’s reagent 1.0 1.0 1.0

7.5% Na2CO3 0.8 0.8 0.8

Incubate at 30 °C for 1.5 h. and take absorbance at 765 nm.

3.2.23.3 ESTIMATION OF TOTAL STEROLS

(Sabir et al., 2003)

Total 5 ml of sample was added to 5 ml of chloroform (1:1). The organic phase (1ml) was

separated and used for estimation of total sterols.

Standard stigmasterol solution: 10 mg of standard stigmasterol was dissolved in 10 ml

chloroform and mixed well. Range of standard: 10-100 µg/ml.

Libermann-Burchard reagent (LB Reagent): 0.5 ml of sulfuric acid cholesterol is

dissolved in 10 ml of acetic anhydride. Covered and kept in ice bucket

BLANK STD SAMPLE

Chloroform 1.0 1.0 -

LB Reagent 1.0 1.0 1.0

Incubate at room temperature for 15’ & take absorbance at 625 nm.

3.2.24 THIN LAYER CHROMATOGRAPHY (TLC)

1) Preparation of GLASS TLC plates:

Take silica gel and make approx 50% solution with d/w

Clean TLC glass plates with methanol and pour the silica gel slowly



97

homogeneously.

Allow to dry the plate at RT.

Before run the plate keep the TLC plate in oven at high temperature.

2) Determine an appropriate solvent system: Various compounds will travel different

distances up the plate depending on the solvent have chosen. In non-polar solvents like

pentane and hexane, most polar compounds will not move, while non-polar compounds

will travel some distance up the plate. In contrast, polar solvents will usually move non-

polar compounds to the solvent front and push the polar compounds off of the baseline. A

good solvent system is one that moves all components of your mixture off the baseline,

but does not put anything on the solvent front – Rf values between 0.15 and 0.85.

Polarity Solubility

Very Polar Methanol > Ethanol > Isopropanol

Moderately Polar Acetonitrile > Ethyl Acetate > Chloroform > Dichloromethane >

Diethyl Ether > Toluene

Non-polar Cyclohexane > Petroleum Ether > Hexane > Pentane

3) Common solvent combinations:

Ethyl Acetate/Hexane: 0–30% most popular combination, sometimes tough to remove

solvents completely on rotary evaporator

Ethanol/Hexane or Pentane: 5–30% useful for very polar compounds

Dichloromethane/Hexane or Pentane: 5–30% sometimes useful when other mixtures fail

4) Fill TLC chamber with 1–2 mL of the desired solvent system. Place a large piece of

cut filter paper in the chamber as well. .

5) Run the TLC. Let the solvent go about 90% of the way up the plate.

6) Remove the plate from the chamber and mark the solvent front immediately

with a pencil and calculate the Rf value.

7) Let the solvent dry off of the plate. .

8) Visualize the TLC using non-destructive technique(s): The best non-destructive

method is the UV lamp wherein keep the plate under the UV lamp and circle any UV

active spots with pencil.



98

3.2.25 HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC)

3.2.25.1 INSTRUMENTATION

Chromatographic separation was achieved on HPTLC plates precoated with silica gel 60

F254 (E. Merck) of 0.2 mm thickness with aluminium sheet support. Samples were spotted

using CAMAG Linomat IV Automatic Sample Spotter (Camag Muttenz, Switzerland)

equipped with syringe (Hamilton, 100 µL). Plates were developed in a glass twin trough

chamber (CAMAG, 20 X 10 cm) pre-saturated with the mobile phase for 30 minutes

Scanning device used was CAMAG TLC Scanner 2 equipped with WinCATS 3 software.

The experimental condition was maintained at 22 ± 2° C.

3.2.25.2 CHROMATOGRAPHIC CONDITION

Parameters Description

Stationary Phase Merck Silica gel 60 F254 HPTLC pre-coated plates

Mode of separation Normal phase

Mobile phase Toluene: Ethyl acetate: Methanol (7.5:5:0.5)

Development chamber Camag twin trough chamber

Chamber saturation 30 minutes

Sample applicator Camag Linomat IV

Band width 10 mm

Space between the bands 10 mm

Rate of sample application 15 secs/µL

Development distance 90 mm

Densitometric scanner Camag Scanner II with WinCats 3 Software

Lamp Tungsten

Wavelength 550 nm

Pre-coated silica gel on aluminum plate 60 F 254, (10 cm ×10 cm, prewashed by methanol

and activated at 60° C for 5 min prior to chromatography)

3.2.25.3 SAMPLE PREPARATION

Aloe vera gel (100 mL) was extracted with 100 ml of - Methanol by mixing - vigorously.

The mixture was kept in a shaker - for 24 hrs -. The organic phase was separated and



99

concentrated to 1 ml with methanol. This concentrated extract was analyzed using

HPLTC.

3.2.25.4 PREPARATION OF STANDARD WORKING SOLUTIONS FOR ASSAY

Preparation of Standard Stock Solutions: Standard Stock solution of Lupeol (1000

µg/mL) was prepared by dissolving accurately weighed standard (10 mg) -in - methanol

(10 mL). From the -standard stock solution, calibrant and quality control samples were

prepared in methanol. Six different calibrant samples: 2, 4, 6, 10, 15, 25 μg/mL,

respectively.

Three quality control samples: 3, 8, 20 µg/mL, respectively

3.2.25.5 SAMPLE APPLICATION

The standard and Aloe extract sample were spotted on pre-coated TLC plates in the form

of narrow bands (6 mm). Samples were applied under continuous drying stream of

nitrogen gas at constant application rate of 150 nL/s.

3.2.25.6 METHOD DEVELOPMENT

After completion of TLC run, plate was developed by 10% Methanolic sulphuric acid

(H2SO4) wherein plate was dipped in solution and kept for 10 seconds. Then, allowed it

to air dry and kept in oven at high heat for 10 minutes. The developed bands were

observed in visible light.

3.2.25.7 METHOD VALIDATION

Linearity and Calibration Curve:

Linearity of the method was determined by analyzing six different calibrant samples

using HPTLC. Calibration curves were plotted over a concentration range of 2-25 µg/mL

by plotting the respective peak area values versus concentrations (n = 6).

Photodocumentation was carried out using Camag Reprostar III, before derivatization

under UV Light 254 nm, 366 nm and after derivatization (Fig.1). The plate was

derivatized with Anisaldehyde‐Sulphuric Acid reagent followed by heating at 110 °C for

10 minutes. The dried plate was scannedimmediately using Camag TLC Scanner III at

wavelength 455 nm. WinCats an integrated Software 4.02 was used for the detection as

well as for the evaluation of data. It was observed that Lupeol appeared at Rf. 0.64

(brown colour).



100

Limit of detection and Limit of quantitation:

The limit of detection (LOD) and limit of quantitation (LOQ) were determined using the

following formulae. LOD = 3 (SD)/S, LOQ = 10 (SD)/S Where, SD = Standard

Deviation of response

Precision:

Precision was evaluated in terms of Intraday and Interday precisions. Intraday precision

was determined by analyzing quality control samples of Lupeol (n=3) at three levels

covering low, medium, and higher concentrations (3, 8, 20 µg/mL) on the same day.

Interday precision was determined by analyzing standard solutions of Lupeol over a

period of three days (n = 3). The peak areas obtained were used to calculate mean and

%RSD (relative SD) values.

Accuracy (%Recovery):

Accuracy of the method was evaluated by carrying out the recovery study at three levels.

Recovery experiments were performed by adding three different amounts of standard

lupeol, that is, 80%, 100%, and 120% of the drug, to the pre-analyzed Aloe extract and

the resultant was reanalyzed three times.

3.2.26 HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)

3.2.26.1 CHROMATOGRAPHIC CONDITION FOR HPLC:

INSTRUMENT

Jasco HPLC-PU 980 pump fitted with AS-1555-10 Auto-

sampler

Mobile Phase Acetonitrile : Ethanol 50:50 (v/v)

Flow-rate 1.0 mL / minute.

Injection Volume 20 µL.

Analytical Column Cosmosil C18 (150 mm x 4.6 mm) Type: Waters

Detector Jasco UV– 970, UV-Visible

Wavelength 210 nm

Integrator Borwin Integrator Software, version 1.21.



101

3.2.26.2 PREPARATION OF STANDARD AND SAMPLE SOLUTION

Standard stock solution of β-Sitosterol, Stigmasterol and Lupeol (1000 µg/mL

each) were prepared by mixing the accurately weighed standards (10 mg each) in

Methanol (10 mL). Working solution of the standards (10 µg/mL) was further prepared

using methanol as a diluents. The petroleum ether extracts of Aloe vera gel was

evaporated and reconstituted in 1 ml of methanol and filtered through 0.45 µm nylon

membrane filter paper.


The standards and sample (20 µL each) were injected into the HPLC system and the run

length was kept constant runtime (30 minutes) while the mobile phase was delivered at -

1ml/min -.

3.2.27 GAS CHROMATOGRAPHY AND MASS SPECTROSCOPY (GC-MS)

3.2.27.1 INSTRUMENTATION

Analyzer: Quadrupole with prefilter

Mass Range: 20-620 Daltons (amu)

Mass Stability: ± 0.1 m/z mass accuracy over 48 hours

Ionization Modes: EI, Positive / Negative Chemical Ionization.

Vacuum Pump: Turbo molecular pump 250L/Sec.

Software: Turbo Mass

Volatile compound from the peel of Aloe vera (L.) was analyzed AutoSystem XL Gas

Chromatograph and the TurboMass Mass Spectrometer under the following condition:

column used were PE-JMS [Column: 30 m x 0.250 mm x 1.0 µm PE-1]. The initial

column temperature was 75 °C and final temperature was 220 °C (5°C/minute), while the

injector temperature was 250 °C with split mode injector and split ratio of 72.6 and

pressure of 14.0 kPa. The flow rate was 1 mL/minute and the flow within the column was

0.50 mL/minute. The detector temperature was 300 ° C and using Helium as the carrier

gas with EI (Electron Impact). Detection: PerkinElmer TurboMass MS.


Samples (1 µL) were injected in to the GC-MS/MS system and the compounds were

identified by comparing their respective retention indices or mass spectra of - with those

of the authentic samples or library.



102

3.2.27.3 CHARACTERIZATION OF UNKNOWN COMPOUND

Interpretation on mass spectra of GC-MS/MS was conducted using the database

of National Institute of Standard and Technology (NIST). The name, molecular weight

and structure of the components of the test samples recorded.

3.3 REFERENCES

Anson, M.L. (1937). The estimation of cathepsin with hemoglobin and the partial purification of cathepsin.

The Journal of general physiology 20, 565-574.

Bonsnes, R.W., and Taussky, H.H. (1945). On the colorimetric determination of creatinine by the Jaffe

reaction. Journal of Biological Chemistry 158, 581-591.

Bowers, G.N., and McComb, R.B. (1975). Measurement of total alkaline phosphatase activity in human

serum. Clinical chemistry 21, 1988-1995.

Buchanan, T.A., Sipos, G.F., Gadalah, S., Yip, K.-P., Marsh, D.J., Hsueh, W., and Bergman, R.N. (1991).

Glucose tolerance and insulin action in rats with renovascular hypertension. Hypertension 18, 341-

347.

Campbell, K.L. (1979). Ovarian granulosa cells isolated with EGTA and hypertonic sucrose: cellular

integrity and function. Biology of reproduction 21, 773-786.

Gorski, J.P., and Kasper, C.B. (1977). Purification and properties of microsomal UDP-

glucuronosyltransferase from rat liver. Journal of Biological Chemistry 252, 1336-1343.

Habig, W.H., Pabst, M.J., and Jakoby, W.B. (1974). Glutathione S-transferases the first enzymatic step in

mercapturic acid formation. Journal of biological Chemistry 249, 7130-7139.

Kafali, H., Iriadam, M., Ozardalı, I., and Demir, N. (2004). Letrozole-induced polycystic ovaries in the rat:

a new model for cystic ovarian disease. Archives of medical research 35, 103-108.

Lowry, O., Farr, A., Rosebrough, N., and Randall, R.J. (1951). Protein measurement of emulsifying

capacity by electrical resistance. J Food Sci 193, 265-275.

Reitman, S., and Frankel, S. (1957). A colorimetric method for the determination of serum glutamic

oxalacetic and glutamic pyruvic transminases.

Sabir, S.M., Hayat, I., and Gardezi, S.D.A. (2003). Estimation of sterols in edible fats and oils. Pak J Nutr

2, 178-181.

Shivanandappa, T., and Venkatesh, S. (1997). A Colorimetric Assay Method for 3β-Hydroxy-Δ 5-steroid

Dehydrogenase. Analytical biochemistry 254, 57-61.

Slinkard, K., and Singleton, V.L. (1977). Total phenol analysis: automation and comparison with manual

methods. American Journal of Enology and Viticulture 28, 49-55.

Zheng, W., and Wang, S.Y. (2001). Antioxidant activity and phenolic compounds in selected herbs. Journal

of Agricultural and Food chemistry 49, 5165-5170.

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